Progress in non-A,non-B hepatitis (NANBH) has been rapid since the successful cDNA cloning of the hepatitis C virus (HCV) genome by Houghton et al. at Chiron. We have developed a sensitive PCR based method to detect the viral genome in clinical samples. This assay seems to be about 10 fold more sensitive that chimpanzee infectivity. We have demonstrated its usefulness by testing sera from 40 patients with chronic NANBH. Interestingly, only 16 of patients 36 of whom were positive for anti-HCV by the Chiron assay had HCV RNA in their serum as measured by PCR. As the sensitivity of the assay was shown to be very high, it was not understood why the positivity rate was so low. Cloning and sequencing has revealed a very high degree of sequence diversity between strains of HCV. Using multiple primer sets we have now detected HCV RNA in 35 of these 40 patients including all 4 who had no detectible antibody with the commercial test. Recently we have shown that the 5' non-coding region of the HCV RNA genome is highly conserved between strains. we have now prepared PCR primers from this region that appear to be universal. We have cloned and sequenced major portions of the genome of HCV including approximately 2000 bases representing the 5' terminus of the genome that had not previously been published (it has now been published in Japan). This is the coding region for the structural genes of HCV. This region seems to be quite unique and is much shorter than the same region in typical flaviviruses. We have initiated expression experiments from this region with the hope of producing useful antigens. We are expressing the putative protease region of the genome with Charles Rice in order to study the processing of the virion polyprotein and the structure and function of the enzyme itself. We are developing possible tissue culture systems in collaboration with Harry Greenberg at Stanford and Curtis Harris at LCI. These experiments are based on human fetal liver and on continuous adult human and chimpanzee cell lines.